Pro-apoptotic Proteins Bid Research Articles

Abstract 18619: Scaffold Protein CARD11 Modulates Receptor Mediated Apoptosis in Cardiomyocytes Following Myocardial Infarction

Background: The CARD scaffold family members, named for containing the common caspase-recruitment domain, are important modulators of inflammatory and apoptotic signalling. CARD11, a membrane bound scaffold, is essential for the production of cytokines in the immune cellular response but is present in heart and lung tissue, where its effects are unknown, and described to associate with apoptotic players such as BCL10 and caspase-8. Due to the complex nature of cardiac response to myocardial infarction (MI), we hypothesize that CARD11 can act as a modulator and integrator of receptor mediated apoptotic pathways following cardiac tissue injury. Methods & Results: CARD11 expression was increased in neonatal cardiomyocytes by general stress induction by H2O2 (25μM) and death receptor activation by Fas agonist Jo2 (1μg/ml) and TNF-alpha (50ng/ml) in isolated neonatal cardiomyocytes. Annexin V (AV) and propidium iodide (PI) double staining demonstrated decreased levels of apoptotic cells (AV+/PI+) following H2O2, Jo2 and TNF-alpha treatments in CARD11-/- cardiomyocytes to that of placebo levels compared to CARD11+/+ cells, as analyzed by flow cytometry. Pretreatment with caspase inhibitor zVAD-FMK (25μM) blocked this increase in apoptosis. Replacement of CARD11 through plasmid transfection restored the apoptotic phenotype in CARD11-/- cells and it was susceptible to zVAD-FMK inhibition. Experimental MI was created in CARD11-deficient mice (CARD11-/-) and wild-type littermate controls (CARD11+/+) by left coronary artery ligation. Survival over 28 days was significantly improved in CARD11-/- mice which exhibited lower rates of rupture and reduced infarct size post MI. TUNEL staining identified a lower percentage of apoptotic cells in the border zone of CARD11-/- hearts post-MI. Western blot analysis displayed decreased levels of cleaved caspase-3, -8 and -9, as well as pro-apoptotic mitochondrial proteins Apaf1, cytochrome-C and Bid. Conclusion: CARD11 up-regulation provides a scaffold for the formation of death receptor signaling through caspase-8 to promote cell death via apoptosis and its removal to increased cardiomyocyte survival in the MI environment.

Synthesis, characterization, DNA interaction, antioxidant and anticancer activity studies of ruthenium(II) polypyridyl complexes

Two new Ru(II) polypyridyl complexes [Ru(phen)2(adppz)](ClO4)2 (1) and [Ru(dip)2(adppz)](ClO4)2 (2) have been synthesized and characterized. The DNA-binding constants were determined to be 6.54±0.42×105 and 7.65±0.20×105M−1 for complexes 1 and 2. DNA binding experiments indicated that complexes 1 and 2 interact with DNA through intercalative mode. Antioxidant activity shows that the complexes have significant hydroxyl radical scavenging activity. Cytotoxic activities suggest that the complex 2 exhibits higher cytotoxic activity against BEL-7402, MG-63 and SKBR-3 cells than complex 1 under identical conditions. Complexes 1 and 2 can induce apoptosis of BEl-7402 cells. We have identified several cellular mechanisms induced by 1 and 2 in BEL-7402 cells, including the level detection of ROS, activation of procaspase 3, caspase 7, the expression of antiapoptotic proteins Bcl-x, Bcl-2, proapoptotic proteins Bad, Bax, Bid and cell cycle arrest. Thus, complexes 1 and 2 inhibit growth of BEL-7402 cells through induction of apoptotic cell death, enhancement of ROS levels and S-phase and G0/G1 cell cycle arrest. Further investigations have shown that complex 2 induces apoptosis by regulating the expression of Bcl-2 family proteins.

Synergistic Induction of Apoptosis in Multiple Myeloma Cells by Bortezomib and Hypoxia-Activated Prodrug TH-302, In Vivo and In Vitro

Recently, we showed that hypoxia is a critical microenvironmental factor in multiple myeloma, and that the hypoxia-activated prodrug TH-302 selectively targets hypoxic multiple myeloma cells and improves multiple disease parameters in vivo. To explore approaches for sensitizing multiple myeloma cells to TH-302, we evaluated in this study the antitumor effect of TH-302 in combination with the clinically used proteasome inhibitor bortezomib. First, we show that TH-302 and bortezomib synergistically induce apoptosis in multiple myeloma cell lines in vitro. Second, we confirm that this synergism is related to the activation of caspase cascades and is mediated by changes of Bcl-2 family proteins. The combination treatment induces enhanced cleavage of caspase-3/8/9 and PARP, and therefore triggers apoptosis and enhances the cleavage of proapoptotic BH3-only protein BAD and BID as well as the antiapoptotic protein Mcl-1. In particular, TH-302 can abrogate the accumulation of antiapoptotic Mcl-1 induced by bortezomib, and decreases the expression of the prosurvival proteins Bcl-2 and Bcl-xL. Furthermore, we found that the induction of the proapoptotic BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA is associated with this synergism. In response to the genotoxic and endoplasmic reticulum stresses by TH-302 and bortezomib, the expression of PUMA and NOXA were upregulated in p53-dependent and -independent manners. Finally, in the murine 5T33MMvv model, we showed that the combination of TH-302 and bortezomib can improve multiple disease parameters and significantly prolong the survival of diseased mice. In conclusion, our studies provide a rationale for clinical evaluation of the combination of TH-302 and bortezomib in patients with multiple myeloma.

Midazolam induces cellular apoptosis in human cancer cells and inhibits tumor growth in xenograft mice.

Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosisinducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-XL and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties.

Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity

In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced a caspase-dependent apoptotic response, being activation of caspase-8, -9 and -3 the result of a post-mitochondrial event.

Histone Deacetylase Inhibitor Upregulates Peroxisomal Fatty Acid Oxidation and Inhibits Apoptotic Cell Death in Abcd1-Deficient Glial Cells

In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.

Effect of sorafenib on cathepsin B-dependent BID-mediated apoptosis in cancer cells.

e15515 Background: Several tyrosine kinase inhibitors (TKIs), have been developed and approved for clinical use in multi-targeted cancer therapy. Among these, sorafenib is an orally available multikinase inhibitor approved for the treatment of the advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Aim of our study was to evaluate the mechanisms responsible for the cytotoxic effects induced by in vitro use of µM doses of sorafenib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with the anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sorafenib markedly reduced at µM dose the viability of BC cells. Treatment for 24h with 20µM of sorafenib, triggered “Incomplete autophagy”, that induced apoptosis of BC cells. Sorafenib by inducing an increased cathepsin B activity and pro-apoptotic protein BID activation, triggered a ROS-mediated-mitochondrial-dependent apoptosis of BC cells. Moreover, the increase of cathepsin B activity induced by sorafenib was inhibited by a specific tyrosine phosphatase inhibitor (e.g., orthovanadate) strongly suggesting for a contribute of tyrosine-phosphatases in sorafenib-induced apoptosis. Conclusions: Sorafenib by triggering incomplete autophagy, stimulates a cathepsin B-induced-BID-mediated-ROS- and mitochondrial-dependent apoptosis of BC cells, which is likely regulated by tyrosine-phosphatases.

Role of Bovine Bcl2A1 Gene in Staurosporine-Induced Apoptotic Cell Lines

Bcl2A1 (B-cell lymphoma 2 - related protein A1) is a member of a gene set that plays a critical role in regulating apoptosis of mammalian cells. The family is divided into 2 groups: (1) anti-apoptotic genes Bcl-2, Bcl-xL, Bcl2A1 and (2) pro-apoptotic genes Bid, Bax, Bak. Apoptosis in cells is regulated by a balanced expression of these groups. In this study, the ORF encoding the 175 amino acid bovine Bcl2A1 protein was amplified by reverse transcription polymerase chain reaction (RT-PCR), using specific primers deduced from the bovine Bcl2A1 mRNA sequence (www//ncbi.nlm.nih.org. Acc: AB195549) and which also contained appropriate restriction endonuclease cleavage sites for cloning. The product was then cloned into plasmid pcDNA3+ to construct the eucaryotic expression plasmid pcDNA3.Bcl2A1. Hela, L11, Vero and WSL cell lines were used to investigate apoptosis induced by staurosporine. Monitoring of cellular DNA fragmentation revealed that incubation with staurosporine at a concentration of 2 µM results in a sufficient level of apoptosis induction in Hela, L11, WSL cell cultures after 6 hours and in Vero cell cultures after 12 hours. Transfection of these cell lines with pcDNA3.Bcl2A1, using pseudorabies virus Us3 protein kinase (PrVUs3) which prevents staurosporine induced apoptosis by interacting with the pro-apoptotic proteins Bid and Bad as a control, proved that expression of the bovine Bcl2A1 gene blocks staurosporine-induced apoptosis in Hela and L11 cell lines. However, this activity was not observed in WSL and Vero cell cultures.

Luteolin inhibits proliferation of human glioblastoma cells via induction of cell cycle arrest and apoptosis

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with limited therapeutic options, and high recurrence and mortality rates. In this study, the anti-tumorigenic properties of luteolin on GBM were examined in vitro. Luteolin was isolated from the whole plant of Glossogyne tenuifolia and the chemical structure was determined by its spectroscopic data. The inhibitory effect of luteolin on the proliferation of GBM 8401 and U87 human glioblastoma cells and the apoptotic effect of this compound on GBM 8401 cells were investigated. Luteolin inhibited the growth of GBM 8401 and U87 cells in a dose- and time-dependent manner. It significantly induced S and G2/M phase cell cycle arrest and apoptotic cell death by decreasing the expression of cyclin-dependent kinase (CDK1), cyclin B1, and anti-apoptotic proteins (Bcl-2, Mcl-1). In addition, luteolin increased the expression of pro-apoptotic proteins (Bid, Bak, Bax, Bad) and activated caspase-3, with a concomitant increased in the levels of cleaved poly-ADP-ribose polymerase (PARP). Moreover, luteolin reduced the cell migration of GBM 8401 cells. Combination treatment of luteolin with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an anticancer drug used in clinical GBM treatment, enhanced significantly the cytotoxic effect of BCNU in GBM 8401 cells. These results suggest that luteolin may be a potential candidate for GBM adjuvant therapy.

Protective effects of onion-derived quercetin on glutamate-mediated hippocampal neuronal cell death

Background:Neurodegenerative diseases are characterized by progressive neuron degeneration in specific functional systems of the central or peripheral nervous system. This study investigated the protective effects of quercetin isolated from onion on neuronal cells and its protective mechanisms against glutamate-induced apoptosis in HT22 cells.Materials and Methods:HT22 cells were cultured to study the neuroprotective mechanism of quercetin against glutamate-mediated oxidative stress. The intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential (ΔΨm) were measured. The protein expression of calpain, spectrin, Bcl-2, Bax, Bid, cytochrome c, and mitogen-activated protein kinases (MAPKs) was evaluated by Western blotting.Results:Quercetin had a protective effect by reducing both intracellular ROS overproduction and glutamate-mediated Ca2+ influx. These effects were due to the downregulation of several apoptosis-related biochemical markers. Calpain expression was reduced and spectrin cleavage was inhibited by quercetin in glutamate-exposed HT22 cells. Disruption of the mitochondrial membrane potential (ΔΨm), activation of the pro-apoptotic proteins Bid and Bax, and cytochrome c release in response to glutamate-induced oxidative stress were reduced. Quercetin also suppressed phosphorylation of MAPKs.Conclusion:This is the first report on the detailed mechanisms of the protective effect of quercetin on HT22 cells. Onion extract and quercetin may be useful for preventing or treating neurodegenerative disorders.

TRAIL enhances paracetamol-induced liver sinusoidal endothelial cell death in a Bim- and Bid-dependent manner

Paracetamol (acetaminophen, APAP) is a universally used analgesic and antipyretic agent. Considered safe at therapeutic doses, overdoses cause acute liver damage characterized by centrilobular hepatic necrosis. One of the major clinical problems of paracetamol-induced liver disease is the development of hemorrhagic alterations. Although hepatocytes represent the main target of the cytotoxic effect of paracetamol overdose, perturbations within the endothelium involving morphological changes of liver sinusoidal endothelial cells (LSECs) have also been described in paracetamol-induced liver disease. Recently, we have shown that paracetamol-induced liver damage is synergistically enhanced by the TRAIL signaling pathway. As LSECs are constantly exposed to activated immune cells expressing death ligands, including TRAIL, we investigated the effect of TRAIL on paracetamol-induced LSEC death. We here demonstrate for the first time that TRAIL strongly enhances paracetamol-mediated LSEC death with typical features of apoptosis. Inhibition of caspases using specific inhibitors resulted in a strong reduction of cell death. TRAIL appears to enhance paracetamol-induced LSEC death via the activation of the pro-apoptotic BH3-only proteins Bid and Bim, which initiate the mitochondrial apoptotic pathway. Taken together this study shows that the liver endothelial layer, mainly LSECs, represent a direct target of the cytotoxic effect of paracetamol and that activation of TRAIL receptor synergistically enhances paracetamol-induced LSEC death via the mitochondrial apoptotic pathway. TRAIL-mediated acceleration of paracetamol-induced cell death may thus contribute to the pathogenesis of paracetamol-induced liver damage.

Relationship between helix stability and binding affinities: molecular dynamics simulations of Bfl-1/A1-binding pro-apoptotic BH3 peptide helices in explicit solvent

The anti-apoptotic protein Bfl-1, also known as A1, belongs to the Bcl-2 family of proteins and interacts with pro-apoptotic Bcl-2 counterparts to regulate programed cell death. As demonstrated for other anti-apoptotic Bcl-2 proteins, Bfl-1/A1 has also been shown to be overexpressed in various human cancers and hence they are attractive targets for anticancer drugs. Peptides derived from the BH3 region of pro-apoptotic Bcl-2 proteins have been shown to elicit similar biological response as that of parent proteins. BH3 peptides from different pro-apoptotic proteins have wide range of affinities for Bfl-1/A1. Experimentally determined complex structures show that the hydrophobic side of amphipathic BH3 peptides binds to the hydrophobic groove formed by the α-helical bundle of Bfl-1/A1 protein. Apart from the length and amino acid composition, a BH3 peptide’s ability to form a stable helical structure has been suggested to be important for its high binding affinity. Molecular dynamics simulations of three BH3 peptides derived from the pro-apoptotic proteins Bak, Bid, and Bmf were carried out each for a period of at least 100 ns after 2 ns equilibration run. The length of simulated BH3 peptides varied from 22 to 24 residues and their binding affinities for Bfl-1/A1 varied from 1 to 180 nM. Our results show that the hydrophobic residues from the hydrophobic face of BH3 peptides tend to cluster together quickly to avoid being exposed to the solvent. This resulted in either reduction of helix length or complete loss of helical character. Bak and Bid BH3 peptides with high affinities for Bf1-1/A1 have stable helical segments in the N-terminal region. The highly conserved Leu residue lies just outside the helical region at the C-terminal end. Capping interactions arising out of N-cap residues seem to be extremely important to maintain the helical stability. Favorable hydrophilic interactions between residues also give further stability to the helix fragment and at least one of the interacting residues resides within the helical region. Bmf BH3 peptide with a weaker binding affinity for Bmf-1/A1 completely lost its helical character at the end of 100 ns production run and a further 50 ns simulation showed that the Bmf peptide continues to remain in random conformation. The present study clearly establishes a link between a BH3 peptide’s ability to form a stable helical segment and its high binding affinity for an anti-apoptotic protein. To further test this hypothesis, we simulated a mutant Bmf peptide for 100 ns in which two residues R129 and H146 were substituted by Asn in silico in the wild-type peptide. Introduction of N-terminal Asn clearly enabled the formation of capping interactions at the N-terminus and resulted in a stable N-terminal helical segment. This demonstrates that the knowledge of interactions that help to maintain stable helical segments in a high-affinity BH3 peptide will help in designing highly specific peptide-based drugs/inhibitors. Such molecules will have the ability to bind a particular anti-apoptotic protein with high affinity.

Mechanisms of Methicillin-Resistant Staphylococcus aureus Pneumonia–Induced Intestinal Epithelial Apoptosis

Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus-induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid-/- mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand-/- mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant.

Endosomes and lysosomes are involved in early steps of Tl(III)-mediated apoptosis in rat pheochromocytoma (PC12) cells

The mechanisms that mediate thallium (Tl) toxicity are still not completely understood. The exposure of rat pheochromocytoma (PC12) cells to Tl(I) or Tl(III) activates both mitochondrial (Tl(I) and Tl(III)) and extrinsic (Tl(III)) pathways of apoptosis. In this work we evaluated the hypothesis that the effects of Tl(III) may be mediated by the damage to lysosomes, where it might be incorporated following the route of iron uptake. PC12 cells exposed for 3h to 100 μM Tl(III) presented marked endosomal acidification, effect that was absent when cells were incubated in a serum-free medium and that was fully recovered when the latter was supplemented with transferrin. After 6h of incubation the colocalization of cathepsins D and B with the lysosomal marker Lamp-1 was decreased together with an increase in the total activity of the enzymes. A permanent damage to lysosomes after 18h of exposure was evidenced from the impairment of acridine orange uptake. Cathepsin D caused the cleavage of pro-apoptotic protein BID that is involved in the activation of the intrinsic pathway of apoptosis. Supporting that, BID cleavage and the activation of caspase 3 by Tl(III) were fully prevented when cells were preincubated with cathepsin D inhibitor (pepstatin A) and only partially prevented when cathepsin B inhibitor (E64d) was used. None of these inhibitors affected BID cleavage or caspase 3 activation in Tl(I)-treated cells. Together, experimental results support the role of Tl(III) uptake by the acidic cell compartments and their involvement in the early steps of Tl(III)-mediated PC12 cells apoptosis.

Abstract 2565: Delphinidin: A dual tyrosine kinase inhibitor of EGFR and VEGFR-2 reduces cell proliferation and induces apoptosis of non-small-cell lung cancer cells

Abstract Lung cancer has proven difficult to control with conventional therapeutic and surgical approaches, and the prognosis is poor with an overall 5-year survival rate of only 15% for non-small-cell lung cancer (NSCLC) in the USA. Over the last decade, the notions of targeting tyrosine kinase receptors for prevention and treatment of cancers have gained considerable attention. The epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2) have emerged as two clinically validated targets for NSCLC. Dual specific tyrosine kinase receptor inhibitors, which target two different classes of growth factor receptors, represent an elegant way to avoid multiple treatment modalities when used alone and in combination with various anti-signaling agents. The development of dietary agents with combined EGFR/VEGFR-2 inhibitory activities in the same molecule may be a step forward to improve antitumor efficacy and to broaden application possibilities. In our pursuit for identification of non-toxic dietary ingredients for prevention and treatment of cancer, we found that delphinidin, an anthocyanidin, present in pigmented fruits (such as pomegranate, berries, and dark grapes) and vegetables (such as egg plant, tomato, carrot and red onion), is a potent inhibitor of EGFR and VEGFR-2 in NSCLC cells. Employing EGFR and VEGFR-2 over expressing NSCLC cells (NCI-H441 and SK-MES-1); we evaluated the effect of delphinidin on EGFR/VEGFR-2 downstream signaling pathways. Delphinidin (5-60 μM) treatment of both NCI-H441 and SK-MES-1 cells inhibited the (i) activation of PI3K, and (ii) phosphorylation of AKT and mitogen activated protein kinases (such as ERK1/2, JNK1/2 and p38). We next determined whether inhibition of EGFR/VEGFR-2 signaling pathways by delphinidin exhibits growth inhibitory effect. We observed that delphinidin treatment of NCI-H441, SK-MES-1 and A-549 cells resulted in a dose-dependent inhibition of cell growth without having any substantial effect on normal human bronchial epithelial cells as assessed by an MTT assay. Treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 μM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and-9, (iii) downregulation of anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax, Bid and Bak), and (v) decrease expression of PCNA and cyclin D1. In addition, we found that delphinidin was effective in reducing the invasive potential of NSCLC cells. This study identifies an abundant fruits and vegetables based anthocyanidin delphinidin as an effective blocker of EGFR and VEGFR-2 in lung cancer cells. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be useful for the management of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2565. doi:1538-7445.AM2012-2565

Abstract 2012: MAP kinase activation and suppression of anti-apoptotic Bcl-2 family members are associated with induction of apoptotic death in melanoma cells following down-regulation of glutathione S-transferase P1

Abstract Introduction. The multifunctional protein, glutathione S-transferase P1, GSTP1, is frequently overexpressed in malignant melanoma and has been associated with increased tumor growth and resistance to therapy and poor overall patient survival. A major cellular function of GSTP1 is as a regulator of cell signaling mediated through its ability to interact with and regulate key signaling proteins, notably, the MAP kinases. In a previous study, we showed that RNA interference-mediated GSTP1 downregulation in GSTP1-overexpressing melanoma cells decreased cell growth and increased the levels of both spontaneous and drug-induced apoptosis. Here, we examined the molecular mechanisms underlying these observations, particularly, those involving alterations in MAP kinase signaling and pro-and anti-apoptotic members of the Bcl2 family of proteins. Methods. Exponentially growing cells of two human melanoma cell lines A375 and DM6 were infected with a lentiviral vector carrying GSTP1-targeted short hairpin RNA (shRNA) targeting. A heterogeneous stable population was isolated by puromycin selection. Alternatively, GSTP1 was knocked down by transfection of the cells with GSTP1-targeted siRNA. The cells were confirmed as GSTP1-ve by PCR and western blotting. At various time-points post GSTP1 suppression, the cells were analyzed for the level of apoptosis by flow cytometry (FCM) and by measuring the level of caspase 3/7 mediated cleavage of the peptide substrate, Ac-DEVD-AFC. Extracts were prepared from control and GSTP1-knockdown cells and analyzed for total and phosphorylated MAP kinases (P38, ERK, and JNK) and for proteins of the Bcl-2 family. Results. There was a near-complete suppression of GSTP1 transcripts and protein in both melanoma cell lines following shRNA-lentiviral infection or at 72 hrs post-siRNA transfection. Over 7 days, there was a steady increase in the level of apoptosis in the cells with GSTP1 suppression, as evidenced by an increase in the pre-G1 population by FCM or in caspase 3/7 activity. This was associated with a significant increase in the level of phosphorylation of the MAP kinases, Erk1/2 and JNK1/2. Following GSTP1 suppression, we also observed a significant decrease in the pro-survival anti-apoptotic Bcl-2 proteins Mcl-1, Bcl-XL, and Bcl-2 with little to no change in pro-apoptotic Bcl2 proteins Bad, Bim and Bid. Conclusion. These results suggest that GSTP1 is obligate for growth and survival of of GSTP1-expressing melanoma cells and that suppression of GSTP1 expression in these cells induces an apoptotic response that involves activation of the MAP Kinases, ERK and JNK, and suppression of anti-apoptotic Bcl-2 proteins. Supported by NIH grants RO1 CA 153050, RO1CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2012. doi:1538-7445.AM2012-2012

Expression, purification, and characterization of recombinant NOD1 (NLRC1): A NLR family member

NOD1 (NLRC1) is a member of the NLR family of innate immunity proteins, which are important cellular sensors of various pathogens. Deregulated NOD1 signaling is involved in various autoimmune, inflammatory, and allergic diseases, making it a potential target for drug discovery. However, to date, the successful high-yield purification NOD1 protein has not been reported. Here we describe the large-scale expression of recombinant NOD1 protein in non-adherent mammalian cells. One-step immunoaffinity purification was carried out, yielding highly pure protein with excellent yields. Gel-sieve chromatography studies showed that the purified NOD1 protein eluted almost exclusively as a monomer. Addition of the NOD1 ligand (γ-Tri-DAP) stimulated NOD1 protein oligomerization. Using purified NOD1 protein for nucleotide binding studies by the Fluorescence Polarization Assay (FPA) method, we determined that NOD1 binds preferentially to ATP over ADP and AMP or dATP. We also documented that purified NOD1 protein binds directly to purified pro-apoptotic protein Bid, thus extending recent data that have identified Bid as an enhancer of NOD1 signaling. This expression and purification strategy will enable a wide variety of biochemical studies of mechanisms of NOD1 regulation, as well as laying a foundation for future attempts at drug discovery.

Characterization of Cholix Toxin-induced Apoptosis in HeLa Cells

Cholix toxin (Cholix) is a novel ADP-ribosylating cytotoxin produced by Vibrio cholerae, which utilizes eukaryotic elongation factor 2 as a substrate and acts by a mechanism similar to that of diphtheria toxin and Pseudomonas exotoxin A. First it was found that Cholix-treated HeLa cells exhibited caspase-dependent apoptosis, whereas intestinal cells such as Caco-2, HCT116, and RKO did not. Here we investigated Cholix-induced cell death signaling pathways in HeLa cells. Cholix-induced cytochrome c release into cytosol was initiated by specific conformational changes of pro-apoptotic Bak associated with Bax. Silencing of bak/bax genes or bak gene alone using siRNA significantly suppressed cytochrome c release and caspase-7 activation, but not activation of caspases-3 and -9. Although pretreatment with a caspase-8 inhibitor (Z-IETD-FMK) reduced Cholix-induced cytochrome c release and activation of caspases-3, -7, and -9, cytotoxicity was not decreased. Pretreatment with Z-YVAD-FMK, which inhibits caspase-1, -4, and -5, suppressed not only cytochrome c release, activation of caspase-3, -7, -8, or -9, and PARP cleavage, but also cytotoxicity, indicating that caspase-1, -4, and -5 activation is initiated at an early stage of Cholix-induced apoptosis and promotes caspase-8 activation. These results show that the inflammatory caspases (caspase-1, -4, and -5) and caspase-8 are responsible for both mitochondrial signals and other caspase activation. In conclusion, we showed that Cholix-induced caspase activation plays an essential role in generation of apoptotic signals, which are mediated by both mitochondria-dependent and -independent pathways.

Antitumor Activity of Metal-Chelating Compound Dp44mT Is Mediated by Formation of a Redox-Active Copper Complex That Accumulates in Lysosomes

The metal-chelating compound Dp44mT is a di-2-pyridylketone thiosemicarbazone (DpT) which displays potent and selective antitumor activity. This compound is receiving translational attention, but its mechanism is poorly understood. Here, we report that Dp44mT targets lysosome integrity through copper binding. Studies using the lysosomotropic fluorochrome acridine orange established that the copper-Dp44mT complex (Cu[Dp44mT]) disrupted lysosomes. This targeting was confirmed with pepstatin A-BODIPY FL, which showed redistribution of cathepsin D to the cytosol with ensuing cleavage of the proapoptotic BH3 protein Bid. Redox activity of Cu[Dp44mT] caused cellular depletion of glutathione, and lysosomal damage was prevented by cotreatment with the glutathione precursor N-acetylcysteine. Copper binding was essential for the potent antitumor activity of Dp44mT, as coincubation with nontoxic copper chelators markedly attenuated its cytotoxicity. Taken together, our studies show how the lysosomal apoptotic pathway can be selectively activated in cancer cells by sequestration of redox-active copper. Our findings define a novel generalized strategy to selectively target lysosome function for chemotherapeutic intervention against cancer.

The IAP-antagonist ARTS initiates caspase activation upstream of cytochrome C and SMAC/Diablo

ARTS (Sept4_i2) is a pro-apoptotic tumor suppressor protein that functions as an antagonist of X-linked IAP (XIAP) to promote apoptosis. It is generally thought that mitochondrial outer membrane permeabilization (MOMP) occurs before activation of caspases and is required for it. Here, we show that ARTS initiates caspase activation upstream of MOMP. In living cells, ARTS is localized to the mitochondrial outer membrane. In response to apoptotic signals, ARTS translocates rapidly to the cytosol in a caspase-independent manner, where it binds XIAP and promotes caspase activation. This translocation precedes the release of cytochrome C and SMAC/Diablo, and ARTS function is required for the normal timing of MOMP. We also show that ARTS-induced caspase activation leads to cleavage of the pro-apoptotic Bcl-2 family protein Bid, known to promote MOMP. We propose that translocation of ARTS initiates a first wave of caspase activation that can promote MOMP. This leads to the subsequent release of additional mitochondrial factors, including cytochrome C and SMAC/Diablo, which then amplifies the caspase cascade and causes apoptosis.